2 The BIG Questions… How are genes turned on & off in eukaryotes? How do cells with the same genes differentiate to perform completely different, specialized functions?
3 Evolution of gene regulation Prokaryotessingle-celledevolved to grow & divide rapidlymust respond quickly to changes in external environmentexploit transient resourcesGene regulationturn genes on & off rapidlyflexibility & reversibilityadjust levels of enzymes for synthesis & digestionprokaryotes use operons to regulate gene transcription, however eukaryotes do not.since transcription & translation are fairly simultaneous there is little opportunity to regulate gene expression after transcription, so control of genes in prokaryotes really has to be done by turning transcription on or off.
4 Evolution of gene regulation Eukaryotesmulticellularevolved to maintain constant internal conditions while facing changing external conditionshomeostasisregulate body as a wholegrowth & developmentlong term processesspecializationturn on & off large number of genesmust coordinate the body as a whole rather than serve the needs of individual cellsSpecializationeach cell of a multicellular eukaryote expresses only a small fraction of its genesDevelopmentdifferent genes needed at different points in life cycle of an organismafterwards need to be turned off permanentlyContinually responding to organism’s needshomeostasiscells of multicellular organisms must continually turn certain genes on & off in response to signals from their external & internal environment
5 Points of controlThe control of gene expression can occur at any step in the pathway from gene to functional protein1. packing/unpacking DNA2. transcription3. mRNA processing4. mRNA transport5. translation6. protein processing7. protein degradation
6 1. DNA packing How do you fit all that DNA into nucleus? DNA coiling & foldingdouble helixnucleosomeschromatin fiberlooped domainschromosomenucleosomes“beads on a string”1st level of DNA packinghistone proteins have high proportion of positively charged amino acids (arginine & lysine)bind tightly to negatively charged DNAfrom DNA double helix to condensed chromosome
7 Nucleosomes “Beads on a string” 1st level of DNA packing 8 histone moleculesNucleosomes“Beads on a string”1st level of DNA packinghistone proteins8 protein moleculespositively charged amino acidsbind tightly to negatively charged DNADNA packing movie
8 DNA packing as gene control Degree of packing of DNA regulates transcriptiontightly wrapped around histonesno transcriptiongenes turned offheterochromatindarker DNA (H) = tightly packedeuchromatinlighter DNA (E) = loosely packedHE
9 DNA methylation Methylation of DNA blocks transcription factors no transcription genes turned offattachment of methyl groups (–CH3) to cytosineC = cytosinenearly permanent inactivation of genesex. inactivated mammalian X chromosome = Barr body
10 Histone acetylation Acetylation of histones unwinds DNA loosely wrapped around histonesenables transcriptiongenes turned onattachment of acetyl groups (–COCH3) to histonesconformational change in histone proteinstranscription factors have easier access to genes
11 2. Transcription initiation Control regions on DNApromoternearby control sequence on DNAbinding of RNA polymerase & transcription factors“base” rate of transcriptionenhancerdistant control sequences on DNAbinding of activator proteins“enhanced” rate (high level) of transcription
12 Model for Enhancer action Enhancer DNA sequencesdistant control sequencesActivator proteinsbind to enhancer sequence & stimulates transcriptionSilencer proteinsbind to enhancer sequence & block gene transcriptionMuch of molecular biology research is trying to understand this: the regulation of transcription.Silencer proteins are, in essence, blocking the positive effect of activator proteins, preventing high level of transcription.Turning on Gene movie
13 Transcription complex Activator Proteins• regulatory proteins bind to DNA at distant enhancer sites• increase the rate of transcriptionEnhancer Sitesregulatory sites on DNA distant from geneEnhancerActivatorActivatorActivatorCoactivatorBFERNA polymerase IIATFIIDHCoding regionT A T ACore promoterand initiation complexInitiation Complex at Promoter Site binding site of RNA polymerase
14 3. Post-transcriptional control Alternative RNA splicingvariable processing of exons creates a family of proteins
15 4. Regulation of mRNA degradation Life span of mRNA determines amount of protein synthesismRNA can last from hours to weeksRNA processing movie
16 RNA interference NEW! Small interfering RNAs (siRNA) short segments of RNA (21-28 bases)bind to mRNAcreate sections of double-stranded mRNA“death” tag for mRNAtriggers degradation of mRNAcause gene “silencing”post-transcriptional controlturns off gene = no protein producedsiRNA
17 Hot…Hot new topic in biology double-stranded miRNA + siRNA Action of siRNAdicer enzymemRNA for translationsiRNAdouble-stranded miRNA + siRNAbreakdown enzyme(RISC)mRNA degradedfunctionally turns gene off
18 1990s | 2006RNA interference“for their discovery of RNA interference — gene silencing by double-stranded RNA”Andrew FireStanfordCraig MelloU Mass
19 5. Control of translation Block initiation of translation stageregulatory proteins attach to 5' end of mRNAprevent attachment of ribosomal subunits & initiator tRNAblock translation of mRNA to proteinControl of translation movie
20 6-7. Protein processing & degradation folding, cleaving, adding sugar groups, targeting for transportProtein degradationubiquitin taggingproteasome degradationThe cell limits the lifetimes of normal proteins by selective degradation. Many proteins, such as the cyclins involved in regulating the cell cycle, must be relatively short-lived.Protein processing movie
21 Ubiquitin 1980s | 2004 “Death tag” mark unwanted proteins with a label 76 amino acid polypeptide, ubiquitinlabeled proteins are broken down rapidly in "waste disposers"proteasomesSince the molecule was subsequently found in numerous different tissues and organisms – but not in bacteria – it was given the name ubiquitin (from Latin ubique, "everywhere")Aaron CiechanoverIsraelAvram HershkoIsraelIrwin RoseUC Riverside
22 Proteasome Protein-degrading “machine” cell’s waste disposer breaks down any proteins into 7-9 amino acid fragmentscellular recyclingA human cell contains about 30,000 proteasomes: these barrel-formed structures can break down practically all proteins to 7-9-amino-acid-long peptides. The active surface of the proteasome is within the barrel where it is shielded from the rest of the cell. The only way in to the active surface is via the "lock", which recognises polyubiquitinated proteins, denatures them with ATP energy and admits them to the barrel for disassembly once the ubiquitin label has been removed. The peptides formed are released from the other end of the proteasome. Thus the proteasome itself cannot choose proteins; it is chiefly the E3 enzyme that does this by ubiquitin-labelling the right protein for breakdownplay Nobel animation
23 Gene Regulation 7 6 5 4 2 1 4 3 protein processing & degradation 1 & 2. transcription- DNA packing- transcription factors3 & 4. post-transcription- mRNA processing- splicing- 5’ cap & poly-A tail- breakdown by siRNA5. translation- block start of translation6 & 7. post-translation- protein processing- protein degradation54initiation of translationmRNA processing21initiation of transcriptionmRNA protectionmRNA splicing43
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